1. Field of the Invention
The invention relates to the immobilization of enzymes and more particularly relates to immobile, site-specific, restriction endonucleases and their use to recognize and cleave specific sequences of base pairs (palindromes) within the deoxyribonucleic acid duplex.
2. Brief Description of the Prior Art
It is well established that restriction endonucleases are powerful tools in studies of genetic engineering, DNA sequencing and molecular biology. These enzymes, which are obtained by relatively simple biochemical fractionation from bacterial extracts are endowed with the ability to recognize and cleave deoxyribonucleic acids at specific sequences. These sequences (palindromes) are the signals for the sites of catalysis for the particular enzyme. To date there are over ninety known restriction endonucleases.
Prior to my invention, restriction endonucleases were physically added to specific deoxyribonucleic acid samples in vitro. The resulting reaction was then stopped. In large preparations where deoxyribonucleic acid fragments have to be recovered, endonuclease had to be first removed from the reaction mixture before fractionating the deoxyribonucleic acid fragments. Although these procedures are singly not limiting, collectively taken they are a set of tedious steps, each one of which potentially results in the loss of deoxyribonucleic acid substrate (which may be difficult to replace). These problems are magnified when secondary cleavages are to be made. For example, in many cases: (a) there may be a blocking of cleavage sites by proteins from the first crude extract, or (b) inhibition of cleavage by several undetermined factors present in extracts of the first or subsequent digestions.
Using the compounds of my invention, deoxyribonucleic acid to be digested may be incubated with a specific restriction endonuclease and the digest recovered quantitatively with simultaneous removal of the endonuclease as well as other objectionable protein materials. The restriction endonucleases and deoxyribonucleic acid may be incubated together in batches or on a mini-column and the digest fragments readily removed from the restriction endonuclease by centrifugation if batchwise digestion is executed or eluted off the mini-column in low salt buffer. In this manner, deoxyribonucleic acid fragments can be obtained in relatively low salt media, free of protein and ready to be submitted to the next step in the investigator's planned study.